1. Field of the Invention
The present invention relates generally to the field of agricultural biotechnology. More particularly, it concerns methods and compositions for the efficient identification of transgenic plant seeds.
2. Description of Related Art
Many attempts have been made to genetically engineer desired traits into plant genomes by introduction of exogenous genes using genetic engineering techniques. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester et al., 1984), polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985), electroporation of protoplasts (Fromm et al., 1986) and microprojectile bombardment (Klein et al., 1987).
An important aspect of the success achieved in transforming plants has been the ability to select or screen for transformed cells and/or plants. Most of the first successes in plant transformation relied on utilization of selectable markers for identification of transformed cells. Genes which have been used for selection of transformed plant cells include, for example, a neomycin phosphotransferase gene (Potrykus et al., 1985) which provides resistance to kanamycin, paromomycin and G418; a bar gene which codes for bialaphos or phosphinothricine resistance (U.S. Pat. No. 5,550,318); a mutant aroA gene which encodes an altered EPSP synthase protein conferring glyphosate resistance (Hinchee et al., 1988); a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate resistant DHFR gene (Thillet et al., 1988); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; and a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan.
More recently, interest has increased in utilization of screenable markers. One particularly useful screenable marker which has been discovered is the green fluorescent protein (GFP) (Sheen et al., 1995). GFP was first cloned and sequenced from the cnidarian Aequorea victoria (Prasher et al., 1992). Since then, it has been expressed in a variety of organisms, ranging from bacteria (Leff and Leff, 1996), fungi (Spellig et al., 1996), invertebrates (Plautz et al., 1996), vertebrates (Zolotukhin et al., 1996) and plants (Haseloff et al., 1997; Reichel et al., 1996; Sheen et al., 1995; Tian et al., 1997). An important advantage of GFP is that it can be assayed non-destructively. Firefly luciferase represents another useful fluorescent marker which can be assayed non-destructively, although detection requires addition of an exogenous substrate (luciferin) for detection (Ow et al., 1986; Millar et al., 1995). In contrast, the assay of the screenable marker GUS is cytotoxic, and therefore, recovery of live transformed cells is difficult (Jefferson et al., 1987).
One means that has been employed for the utilization of screenable markers, such as GFP, has been the fusion of the screenable marker with a selectable marker. Fusion proteins have been made, for example, between GFP and the selectable marker neomycin phosphotransferase (NPTII) (Genbank Accession No. AF004665), between GFP and hygromycin phosphotransferase (Lybarger et al. 1996), between the firefly luciferase gene (luc) and the neomycin phosphotransferase (NPTII) (Barnes, 1990), and between the ALS gene and GFP or GUS (WO 97/41228). The screening of transgenic maize cells based on GFP expression was described in WO 97/41228.
The identification of transgenic seeds using a GFP screenable marker also was described in WO 97/41228. In this case, however, the GFP gene was constitutively expressed using the ubiquitin promoter. It was found that seeds expressing GFP did not germinate when planted in soil, and could only be germinated by excision of embryos and plating on growth medium.
The failure to identify a method of non-destructively identifying transgenic seeds which can be directly germinated has represented a significant hindrance to breeders during the development of novel transgenic plant lines. Without labor-intensive embryo-rescue, current technology requires the growing and assaying of potentially transgenic seeds. The identification of a direct means for selection of transgenic cells, regeneration of plants from the transgenic cells and the screening of transgenic seeds which could, in turn, be directly advanced in breeding protocols would greatly improve scientists abilities to develop novel transgenic plant lines for the benefit of consumers.